The present invention relates generally to novel polynucleotides and the proteins encoded thereby and more particularly, to polynucleotides encoding a novel mammalian phosphatidylinositol-4-phosphate-5-kinase (PIP5K), and therapeutic, diagnostic and research methods employing same.
Insulin action to recruit an intracellular pool of the glucose transporter protein GLUT4 to the fat/muscle cell surface has been established for more than a decade, yet the molecular details of this phenomenon are still elusive. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995). Intriguingly, while GLUT4 appears to be a unique isoform for fat and muscle tissues, signaling element(s) specifically implicated in its sorting, directing and insulin-sensitive delivery to the cell surface are presently unknown. Phosphatidylinositol 4-phosphate-5-kinase (PIP5K; EC 2.7.1.68) has been implicated in membrane trafficking in yeast. Yamamoto, A. et al., Mol Biol. Cell. 6:525-539 (1995). In particular, PIP5K synthesizes phosphatidylinositol 4,5-bisphosphate (Ptdlns[4,5]P2) from phosphatidylinositol-4-phosphate (Ptdlns[4]P). Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). It has recently been reported that PIP5K also synthesizes Ptdlns[4,5]P2 from phosphatidylinositol-5-phosphate (Ptdlns[5]P). Hinchliffe, K. et al., Nature 390:123-124 (1997). The biosynthesis of Ptdlns[4,5]P2 has attracted increasing interest because of mounting evidence implicating metabolites of Ptdlns[4,5]P2 as important regulators of many cellular processes. Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). In particular, Ptdlns[4,5]P2 is a key substrate of insulin-activated PI 3-kinase, which enzyme, together with its Ptdlns[3,4]P2 and Ptdlns[3,4,5]P3 products, appear to be important elements in insulin action on GLUT4 membrane movements. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995).
The key role of activated PI 3-kinase implies the presence of a large, easily available phosphoinositide substrate pool and suggests that the local production of Ptdlns[4]P and Ptdlns[4,5]P2 lipid substrates at key insulin-sensitive intracellular locations would aid an efficient PI 3-kinase reaction and may be crucial for the PI 3-kinase-mediated effect of insulin in GLUT4 directing and delivery to the fat/muscle cell surface. In addition, an alternative pathway of generating Ptdlns[3,4]P2 and Ptdlns[3,4,5]P3 has been recently suggested which utilizes Ptdlns[3]P substrates and concert action of phosphatidylinositol4-phosphate-5-kinases. Zhang, X. et al., J. Biol. Chem. 272:17756-17761 (1997). Taken together, these data are consistent with the notion that the activity of PIP5K can contribute to the regulated pools of Ptdlns[3,4]P2 and Ptdlns[3,4,5]P3 stimulated by growth factors and insulin.
Two distinct mammalian PIP5Ks, called type I (PIP5KI) and type II (PIP5KII), isolated from bovine and human erythrocytes, respectively, have been reported (Bazenet, C. E. et al., J. Biol. Chem. 265: 18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), as well as an isoform of PIP5KII (PIP5KIIxcex1). Boronenkov, I. V. et al., J. Biol. Chem. 270:2881-2884 (1995). Yeast isozymes, specifically MSS4 and fab 1, have also been isolated and studied. Yamamoto, A. et al., Mol. Biol Cell 6:525-539 (1995); Yoshida, S. et al., Mol. Gen. Genet. 342:631-640 (1994); Yamamoto, A. et al., Mol. Biol Cell 6:525-539 (1995).
As mentioned above, the conversion from Ptdlns[4]P to Ptdlns[4,5]P2 is an important branch point in the phosphoinositide (PI) cycle, depicted in FIG. 1A. FIG. 1B depicts newly described inositol lipids, Ptdlns[5]P and Ptdlns[3,5]P2, and FIG. 1C includes the novel alternative pathway for Ptdlns[3,4,5]P3 production by PIP5Ks. The hydrolysis of Ptdlns[4,5]P2 by phosphoinositide-specific phospholipase C(PLC; EC 3.1.4.3) generates the second messengers, 1,2-diacylglycerol and inositol 1,4,5-triphosphate. 1,2-diacylglycerol activates several protein kinase C isoforms while inositol 1,4,5-triphosphate causes an increase in intracellular calcium. Rana, R. S. Physiol. Rev. 70:115-164 (1990). Ptdlns[4,5]P2 can also be phosphorylated by a PI 3-kinase (EC 2.7.1.137) to phosphatidylinositol 3,4,5-triphosphate (Ptdlns[3,4,5]P3), a second messenger whose targets are largely unknown but may include protein kinase C isoforms. Nakanishi, H. et al., J. Biol. Chem. 268:13-16 (1993); Toker, A. et al., J. Biol. Chem. 269:32358-32367 (1994). Furthermore, Ptdlns[4,5]P2 modulates the function of numerous enzymes including many actin-binding proteins (Janmey, P. A., Annu. Rev. Physiol. 56:169-191 (1994)), binds Ph domains found in some signaling proteins (Harlan, J. E. et al., Nature 371:168-170 (1994)), and appears to be involved in the secretory vesicle cycle. Eberhard, D.A. et al., Biochem. J. 268:15-25 (1990); Hay, J. C. et al., Nature 374:173-177 (1995); Liscovitch M. et al., Cell 81:659-662 (1995).
PIP5Ks have been isolated from erythrocytes, brain, adrenal medulla, liver and other sources. Carpenter, C. L. et al., Biochemistry 29:11147-11156 (1990) (and references therein); Van Dongen, C. J. et al., Biochem. J. 233:859-864 (1986); Moritz, A et al., Biochim. Biophys. Acta 1168:79-86 (1993); Divecha, N. et al., Biochem. J. 288:637-642 (1992); Husebye, E. S. et al., Biochim. Biophys. Acta 1010:250-257 (1989); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990). In cells, PIP5K activity is found on the plasma membrane (Carpenter, C. L. et al., Biochemistly 29:11147-11156 (1990); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990); Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Smith, C. D. et al., J. Biol. Chem. 264:3206-3210 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), associated with the cytoskeleton (Payrastre, B. et al., J. Cell Biol. 115:121-128 (1991); Grondin, P. et al., J. Biol Chem. 266:15705-15709 (1991)), on the endoplasmic reticulum (Helms, J. B. et al., J. Biol Chem. 266:21368-21374 (1991), and in nuclei (Divecha, N. et al., Biochem. J. 289:617-620 (1993); Payrastre, B. et al., J. Biol. Chem. 267:5078-5084 (1992); Divecha, N. et al., Cell 74:405407 (1993)). There is also a soluble, cytosolic population of PIP5K. Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994); Moritz, A. et al., J. Neurochem. 54:351-354 (1990). The kinase""s product, Ptdlns[4,5]P2, is primarily found in the plasma membrane but can be detected in isolated endoplasmic reticulum and nuclei. Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Ptdlns[4]P is present in all of these fractions. Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Hinchliffe, K. et al., Nature 390:123-124 (1997); Rameh, L. E. et al., Nature 390:192-196 (1997).
One postulated reason for the large family of PIP5Ks is that many forms of regulation and cellular functions have been attributed to PIP5Ks, as summarized in FIG. 2. It would thus be desirable to provide a mechanism to further study the role of PIP5Ks. It would also be desirable to provide a novel mammalian PIP5K. It would further be desirable to provide a screening method for further studying the role of PIP5Ks, their substrates and products. It would still further be desirable to provide an animal model for further investigating the role of PIP5Ks.
A novel polynucleotide encoding a mammalian PIP5K referred to herein as p235, is provided. p235 is specifically expressed in adipocytes and myocytes and is believed to be involved in membrane trafficking, particularly, insulin-induced membrane trafficking of fat/muscle specific glucose transporter, GLUT4. The isolated cDNA for p235 set forth in SEQ ID NO: 1 is about 7.4 kbp long with an open reading frame extending from nucleotide 139 to 6294, encoding the novel protein. p235 is 2052 amino acids in length with Mr 233,040 and pl 6.34. The deduced polypeptide sequence is set forth in SEQ ID NO: 2.
Thus, in one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1,
b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2,
c) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2, having biological activity,
d) a polynucleotide which is an allelic variant of the polynucleotide of a) and,
e) a polynucleotide which encodes a species homologue of the protein of b) or c).
In another embodiment, the present invention provides a gene corresponding to the cDNA of SEQ ID NO: 1.
In yet another embodiment, the present invention provides a composition comprising a protein wherein the protein comprises an amino acid sequence selected from the group consisting of:
a) the amino acid sequence of SEQ ID NO: 2, and
b) fragments of the amino acid sequence of SEQ ID NO: 2.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
Methods of using the polynucleotide of the present invention and the protein encoded thereby to further study the role of PIP5Ks, their substrates and products, are also provided as well as research models including cell lines and transgenic and knockout animal models.
Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.